Sensorineural hair cell differentiation

ABSTRACT

The present disclosure provides compositions and methods for treating subjects at risk for or with sensorineural hearing loss. Such compositions and methods include modulating the epigenetic status of the cell, or rate of protein degradation, to increase expression and/or levels of Atoh1 protein.

CLAIM OF PRIORITY

This application is a continuation application of U.S. patentapplication Ser. No. 15/306,657, filed Oct. 25, 2016, which is a 371U.S. National Application of PCT/US2015/028035, filed on Apr. 28, 2015,which claims priority under 35 USC § 119(e) to U.S. Patent ApplicationSer. No. 61/985,170, filed on Apr. 28, 2014, the entire contents ofwhich are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to the generation of sensorineural hair cells,and more particularly to the use of epigenetic modulation of Atoh1expression to generate sensorineural hair cells.

BACKGROUND

There are six distinct sensory organs in the mammalian inner ear: thethree cristae of the semicircular canals, the two maculae of the sacculeand utricle, and the organ of Corti of the cochlea. The organ of Cortiis the organ of hearing. The receptor cell for hearing is the hair cellof the cochlea (referred to herein as a hair cell, a sensory hair cell,or a sensorineural hair cell). Hair cells are limited in number and donot regenerate in mammals; damage or death of these cells leads tohearing loss (Edge and Chen, Curr. Opin. Neurobiol., 18:377-382 (2008)).Therapeutic compositions and methods to increase sensorineural hair cellnumber and/or function in the cochlea are required to address hearingloss.

SUMMARY

The present disclosure provides compositions and methods for generatinghair cells and supporting cells by epigenetic modification of cochlearcells. Both cell division and cell differentiation are modified bychanging epigenetic marks. The data presented herein show a role ofSox2-mediated and Wnt-mediated epigenetic modulation of thetranscriptional regulation of Atoh1 and the effects of epigeneticmodulation on expression of genes in the cell cycle and Notch and Wntpathways.

Thus, in a first aspect the invention provides methods for treatingsensorineural hearing loss associated with loss of auditory hair cellsin a subject. The methods include administering to the subject, e.g., tothe inner ear of the subject, a pharmaceutical composition comprisingone or more of the following: a Histone Deacetylase (HDAC) inhibitor; ahistone methyltransferase (HMT) inhibitor; a DNA methyltransferase(DNMT) inhibitor; a Histone Lysine Demethylase (KDM) inhibitor; anR-spondin; activators of c- and n-myc or Wnt agonists; and/or aninhibitory nucleic acid that specifically reduces expression of Hic1.

In another aspect, the invention provides the use for the treatment ofsensorineural hearing loss associated with loss of auditory hair cellsin a subject of pharmaceutical compositions comprising one or more ofthe following: a Histone Deacetylase (HDAC) inhibitor; a histonemethyltransferase (HMT) inhibitor; a DNA methyltransferase (DNMT)inhibitor; a Histone Lysine Demethylase (KDM) inhibitor; an R-spondin;activators of c- and n-myc or Wnt agonists; and/or an inhibitory nucleicacid that specifically reduces expression of Hic1.

In some embodiments, the HDAC inhibitor is selected from the groupconsisting of: Sodium Butyrate, Trichostatin A, hydroxamic acids, cyclictetrapeptides, trapoxin B, depsipeptides, benzamides, electrophilicketones, aliphatic acid compounds, pyroxamide, phenylbutyrate, valproicacid, hydroxamic acids, romidepsin, vorinostat (SAHA), belinostat(PXD101), LAQ824, panobinostat (LBH589), entinostat (MS275), CI-994(N-acetyldinaline, also tacedinaline), Entinostat (SNDX-275; formerlyMS-275), EVP-0334, SRT501, CUDC-101, JNJ-26481585, PCI24781, Givinostat(ITF2357), and mocetinostat (MGCD0103).

In some embodiments, the EZH2/HMT inhibitor is selected from the groupconsisting of Deazaneplanocin A (DZNep), PR-SET7, GSK126, GSK J1,EPZ005687; E7438; EI1; EPZ-6438; GSK343; BIX-01294, UNC0638, BRD4770,EPZ004777, AZ505 and PDB 4e47. In some embodiments, the EZH2/HMTinhibitor is selected from the group consisting of EPZ005687; E7438;EI1; EPZ-6438; GSK343; BIX-01294, UNC0638, BRD4770, EPZ004777, AZ505 andPDB 4e47

In some embodiments, the DNMT inhibitor is selected from the groupconsisting of azacytidine, decitabine, Zebularine(1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one), procainamide,procaine, (−)-epigallocatechin-3-gallate, MG98, hydralazine, RG108, andChlorogenic acid.

In some embodiments, the KDM inhibitor is selected from the groupconsisting of tranylcypromine ((trans-2-phenylcyclopropyl-1-amine,trans-2-PCPA)) and analogs thereof with substitutions at the benzenering; 2,4-pyridinedicarboxylic acid (2,4-PDCA);5-Carboxy-8-hydroxyquinoline (IOX1) and n-octyl ester thereof. In someembodiments, the KDM inhibitor is selected from the group consisting oftranylcypromine ((trans-2-phenylcyclopropyl-1-amine, trans-2-PCPA)) andanalogs thereof with substitutions at the benzene ring;2,4-pyridinedicarboxylic acid (2,4-PDCA); 5-Carboxy-8-hydroxyquinoline(IOX1) n-octyl ester thereof, and Pargyline(N-Methyl-N-propargylbenzylamine).

In some embodiments, the R-spondin is human R-spondin 1 (hRSPO1),hRSPO2, hRSPO3, or hRSPO4.

In some embodiments, the inhibitory nucleic acid that specificallyreduces expression of Hic1 is an siRNA, shRNA, or antisenseoligonucleotide

In some embodiments, the methods include administering thepharmaceutical composition to the subject, e.g., to the inner ear of thesubject.

In some embodiments, the methods include application of thepharmaceutical composition to the round window membrane, e.g.,intra-tympanic injection, or direct delivery into the inner ear fluids,e.g., using a microfluidic device.

In some embodiments, the pharmaceutical composition is formulated foradministration to the inner ear of the subject, e.g., to the roundwindow membrane, e.g., intra-tympanic injection, or direct delivery intothe inner ear fluids, e.g., using a microfluidic device.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1B (A) Cartoons showing the consensus binding sequence for Pax2and Sox2 between positions 303-315 of the Atoh1 3′ enhancer. Additionalbinding sites are shown as boxes. (B) Shown sequence homology betweenhuman Atoh1 (SEQ ID NO:2) and mouse Atoh1 (SEQ ID NO:1) within thebinding region.

FIGS. 2A-D DNA gels resulting from chromatin immunoprecipitation (ChIP)studies using Pax2 or Sox2 antibodies. Chromatin-immunoprecipitation(ChIP) was performed with Pax2 or Sox2 antibodies (3 Mg each) in OC-1cells. Protein complexes with sheared DNA bound to beads were resolved,and PCR was performed. Primers covering the entire length of the Atoh13′ enhancer were used and the bands for the first 4 primers are shown.ChIP with a Pax2 antibody gives 2 bands between 4-375 bp on the Atoh1enhancer (the putative Sox2-Pax2 binding site lies between bases303-335) and a faint band between 446-637 bp, apparently due to thesecond Pax2 binding site in the enhancer. ChIP with a Sox2 antibody withprimer 153-375 yields one band (A). The region of DNA containing thebinding site (153-375) is amplified by primers covering the proposedsite after ChIP using Pax2 or Sox2 but not control (N IgG) antibodies inHEK cells transfected with the Atoh1 3′ enhancer (B). Primer 4×P/Samplified the 4×-binding site construct after immunoprecipitation withPax2 or Sox2 antibodies but not the serum control after ChIP of OC-1cells (input is 1:10 DNA before ChIP) transfected with the construct(C). Addition of siRNA for Pax2 or Sox2 (100 nM) reduced the amount ofDNA precipitated by the Pax2 antibody from HEK cells (input is 1:10 DNAbefore ChIP) transfected with the 4×-binding site construct, Pax2, andSox2 (D).

FIGS. 3A-C Schematic representation of the Atoh1 3′ enhancer reporterconstructs used for luciferase studies. Reporters contained either theintact mouse Atoh1 3′ enhancer (a) or a 4×repeat of the Sox2-Pax2binding site (b). The two mutated reporter constructs (c) based on theconstruct shown in b contained additional bases between the bindingsites (Mutation 1) or had both binding sites mutated (Mutation 2).

FIGS. 4A-C Images of Western blots. Pax2 and Sox2 identified by IP andWestern blotting of cell lysates. After transfection of OC-1 cells withPax2 and Sox2, Western blotting for Sox2 was performed of the proteinsobtained by IP of OC-1 cells with Pax2, Sox2 or control (N IgG)antibodies (a). IP of Pax2 and Sox2 transfected HEK cell lysates wasfollowed by Western blotting for Sox2 or Pax2 (b). IP of HEK chromatinlysate with either Pax2 or Sox2 antibody was followed by Western blotanalysis with either Sox2 or Pax2 antibody. Sox2 antibody precipitatesPax2, and Pax2 antibody precipitates Sox2 (c).

FIG. 5 Bar graph showing Atoh1 reporter activity in IEC6 cells in thepresence of transfected Pax2, Sox2, or both Pax2 and Sox2.

FIG. 6 Bar graph showing Atoh1 reporter activity in IEC6 cells.Luciferase activation of the 4×-binding site construct was assessed withor without exogenous transfection of Pax2 and Sox2 (1 ng each) in IEC6cells. Activation is doubled by co-transfection of Pax2 and Sox2.

FIGS. 7A-B Bar graphs showing Atoh1 reporter activity in IEC6 cellstransfected with 4×-binding site construct and 1 ng of Pax2 and Sox2 andtreated with control siRNA or Pax2 and Sox2 siRNAs (100 nM total) showreduced activation (A). Overexpression of Pax2 (1 ng) and Sox2 (0 ng-100ng) in IEC6 cells transfected with the 4×-binding site construct (100ng). Upregulation is greatest with a combination of 1 ng each Pax2 andSox2 (B).

FIGS. 8A-8C (A) Schematic representation of the intact Atoh1 reporterconstruct used in the reporter assays summarized in (B) and (C). (B)-(C)Bar graphs showing Atoh1 reporter activity in HEK and Neuro2a cellstransfected with Pax2, Sox2, or both Pax2 and Sox2.

FIGS. 9A-D (A) Bar graph showing activity of mutant Atoh1 reporterconstructs. (B) Schematic representation of the mutant Atoh1 reporterconstruct and its activity in IEC2 cells transfected with Sox2, Pax2, orboth (B-D).

FIG. 10 Bar graph showing IEC6 cells transfected with 4×-binding siteconstruct and 1 ng of Pax2 and Sox2 and treated with control siRNA orPax2 and Sox2 siRNAs (100 nM total).

FIGS. 11A-B Bar graphs showing Atoh1 mRNA levels following transfectionof Pax2, Sox2, or both.

FIGS. 12A-B (A) Bar graph showing that the combination of FGF2 and DAPTresults in the greatest increase in the number of myosin VIIa-positivecells as a fraction of the total cells compared to control (*, p<0.05;**, p<0.01). (C) Newly formed hair cells after treatment with DAPT andFGF2 are Atoh1 and myosin VIIa-positive. Scale bar represents 50 μm. (B)Inner ear stem cells under differentiating conditions in the presence ofDAPT and treated with siRNA for Pax2 or Sox2 or both (100 nM total)Counting myosin VIIa-positive cells vs. total number of cells showed areduced number of hair cells. The greatest reduction (*, p<0.05; **,p<0.01) was found in cultures treated with a combination of siRNAs forPax2 and Sox2 (50 nM each).

FIGS. 13A-B Treatment with DAPT and FGF2 results in the greatestincrease in the percentage of Atoh1-GFP positive cells/100 μm (a) and inthe percentage of Atoh1-GFP positive cells that co-express Pax2 and Sox2(b).

FIGS. 14A-B Bar graphs showing changes in relative levels of H3K4Me3,H3K27Me3 and EZH2 at the Atoh1 (14A) and Sox2 (14B) loci.

FIG. 15 Bar graph showing the effect of HDAC inhibitors on Atoh1.Treatment of OC-1 cells with HDAC inhibitors, VPA and TSA, increasedexpression of Atoh1, as measured by qRT-PCR. Values were calculatedrelative to 18S RNA.

FIG. 16A Western blot of HEK cells co-transfected with FLAG-Atoh1 andSox2 and probed after 24 hr with anti-FLAG (upper panel) and anti-Sox2(lower panel) antibodies.

FIG. 16B Bar graph showing Atoh1 mRNA levels by real time qPCR inSox2-transfected OC-1 cells.

FIGS. 17A-B Bar graph showing Atoh1 mRNA levels in cells expressingvarious constructs as indicated.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the surprisingdiscovery that epigenetic modulation results in supporting cell divisionand increases Atoh1 expression, which is expected to increase generationof hair cells and support cells. As shown herein, Sox2 and Pax2 interactwith each other and the three prime (3′) enhancer for Atoh1, atranscription factor required for hair cell differentiation, at acompound consensus sequence, and these interactions lead to hair celldifferentiation. Stimulation of the Wnt pathway also results in celldivision and hair cell differentiation. In addition, modulating theepigenetic state of Atoh1 lead to an increase in Atoh1 expression. Asincreased Atoh1 leads to an increase in generation of hair cells, theseepigenetic modifiers are expected to increase generation of hair cellsfrom progenitors.

It is widely accepted that although cells capable of generating haircells are present in the inner ear, natural hair cell regeneration inthe inner ear is low (Li et al., Trends Mol. Med., 10, 309-315 (2004);Li et al., Nat. Med., 9, 1293-1299 (2003); Rask-Andersen et al., Hear.Res., 203, 180-191 (2005)). As a result, lost or damaged hair cells maynot be adequately replaced by natural physiological processes (e.g.,cell differentiation), and a loss of hair cells occurs. In manyindividuals, such hair cell loss can result in, e.g., sensorineuralhearing loss, hearing impairment, and balance disorders. Therapeuticstrategies that increase the number of hair cells in the inner ear willbenefit a subject with hair cell loss, e.g., with one or more of theseconditions.

In some embodiments, the present disclosure provides compositions andmethods for preventing and/or treating auditory disease in a subject byincreasing the number of sensory epithelial cells of the inner ear(e.g., hair cells and/or support cells), e.g., in the inner ear of thesubject, by administering to the subject (e.g., to the inner ear of thesubject) compositions that modulate the level of Sox2 and Pax2 in targetcells. In other embodiments, the present disclosure providescompositions and methods for preventing and/or treating auditory diseasein a subject by increasing the number of sensory epithelial cells of theinner ear (e.g., hair cells and/or support cells), e.g., in the innerear of the subject, by administering to the subject (e.g., to the innerear of the subject) compositions that modulate the level of β-catenin intarget cells.

Atoh1 is important for specifying hair cell fate and its expression istightly regulated (Kelly et al., Nat. Rev. Neurosci., 7:837-849 (2006)).Once activated, Atoh1 interacts with its 3′ enhancer in a positivefeedback loop to maintain expression (Helms et al., Development, 127:1185-1196 (2000)). Despite the reported importance of Atoh1 for haircell differentiation (Bermingham et al., Science, 284:1837-1841 (1999);Zheng and Gao, Nat. Neurosci., 3:580-586 (2000); Izumikawa et al., Nat.Med., 11:271-276 (2005); Kelly et al., Nat. Rev. Neurosci., 7:837-849(2006); Gubbels et al., Nature, 455:537-541 (2008); Jeon et al., J.Neurosci., 31:8351-8358 (2011)), pathways and factors involved in itsregulation are reportedly poorly understood (Fritzsch et al., Dev. Dyn.,233:570-583 (2005)).

As described herein, targeted modulation of methylation can be used toregulate Atoh1 and promote hair cell differentiation.

Pax2/Sox2

Pax2 is a member of the paired box transcription factor family. The paxgene family encodes transcription factors implicated in the control ofmammalian development and characterized by the presence of a 128 aminoacid DNA-binding domain, referred to as the paired box Gruss ad Walther,69:719-722 (1992); Stuart et al., Ann. Rev. Gen., 28:219-236 91994)). Invertebrates, the nine members of the pax family can be classified infour groups based upon the presence of conserved structural domains,sequence homology and similar expression pattern (Dahl et al.,Bioassays, 19:755-767 (1997); Mansuri et al., J. Cell Sci., 18:35-42(1994)). Pax2, Pax5 and Pax8 form group II, which is characterized bythe presence of a paired domain, an octapeptide motif and a partialpaired-class DNA-binding homeodomain. The pax gene family has been shownto play important roles in the development of various structures andorgans (Zaiko et al., E. J. Endocrinol., 150:389-395 (2004). Absence ofPax2 results in major developmental defects of the central nervoussystem, eyes, ears and urogenital system (Favor et al., PNAS,93:13870-13875 (1996)). Pax2 and its homologues are reportedly involvedin the differentiation of the Drosophila shaft cells (Kavaler,Development, 126:2261-72 (1999)), and in branching morphogenesis andnephron differentiation in the developing kidney (Narlis et al., J. Am.Soc. Nephrol., 18:1121-9 (2007)). Reports also support a role for Pax2in inner ear development and hair cell differentiation (Warchol andRichardson, Dev. Neurobiol., 69:191-202 (2009)). Mutations in Pax2 inhumans result in renal coloboma characterized by kidney, eye, and earabnormalities, including deafness (Schimmenti et al., Am. J. Hum.Genet., 60:869-878 (1997)). Pax2 hereafter refers to Pax2, DNA, MRNA,and protein.

Sox2 is an intronless gene that encodes a member of the SRY-relatedHMG-box (SOX) family of transcription factors involved in the regulationof embryonic development and in the determination of cell fate. The genelies within an intron of another gene called SOX2 overlapping transcript(SOX2OT). Sox2 functions as a transcription factor that forms a trimericcomplex with OCT4 on DNA and controls the expression of a number ofgenes involved in embryonic development such as YES1, FGF4, UTF1 andZFP206 (see, e.g., Avilion et al., (2003); Masui et al., (2007);Takahashi et al., Cell, 131:861-872 (2007)). Sox2 is suggested toinhibit the expression of differentiation factors (Boyer et al., Cell,122:947-956 (2005); Bylund et al., Nat. Neurosci., 6:1162-1168 (2003);Episkopou, Trends Neurosci., 28:219-221 (2005); Graham et al., Neuron,39:749-765 (2003)), and to facilitate pluripotency of stem cells (Boyeret al., Cell, 122:947-956 (2005); Takahashi and Yamanaka, (2006); Yu etal., (2007)). Sox2 is one of four transcription factors known toreprogram various cell types to induced pluripotent stem cells(Yamanaka, Cell. Stem Cell, 1:39-49 (2007)).

In the sensory epithelia of the inner ear, Sox2 is initially expressedin progenitors of both hair cells and support cells (Kiernan et al.,Nature, 434:1031-1035 (2005); Hume et al, Gene Expr. Patterns, 7:798-807(2007); Neves et al., J. Comp. Neurobiol., 503:487-500 (2007)). Itsexpression is ultimately lost from hair cells after differentiation butis maintained in support cells (Kiernan et al., Nature, 434:1031-1035(2005); Hume et al, Gene Expr. Patterns, 7:798-807 (2007); Neves et al.,J. Comp. Neurobiol., 503:487-500 (2007)). Mutations in Sox2 in humansresult in eye defects that can be accompanied by deafness (Kelberman etal., J. Clin. Invest., 116:2442-2455 (2006)). Roles for Sox2 in thedevelopment and/or regeneration of the inner ear are reported (see,e.g., Millima Ki et al., Dev. Biol. 338(2):262 (2010); Kiernan et al.,Nature, 434:1031-1035 (2005). Sox2 expression was reported to reduceAtoh1 expression and antagonize hair cell differentiation (Dabdoub etal., Proc. Natl. Acad. Sci., 105:18396-18401 (2008)). Other reportssuggest a role for Sox2 in hair cell maintenance (Kiernan et al.,Nature, 434:1031-1035 (2005)). Sox2 hereafter refers to Sox2, DNA, MRNAand protein.

Pax2 and Sox2 knockout animals have severe deficiencies in thedeveloping inner ear (Burton et al., Dev. Biol. 272:161-175 (2004);Lawoko-Kerali et al., J. Comp. Neurol., 442:378-391 (2002); Ohyama etal., Development, 133:865-875 (2006); Tones et al., Development122:3381-3391 (1996)) and expression patterns of Sox2 and Pax2 in theinner ear are described in Moreno et al., PLoS One, 5(12):e15907 (2010).

Ezh2

As described herein, Sox2-Pax2 interact with each other and inassociation with other proteins recruit methyltransferases, andβ-catenin interacts with Tcf/Lef family proteins and with Hic-1,resulting in altered methylation and acetylation patterns at the Atoh1enhancer. Sox2 and the Polycomb repressive complex 2 (PRC2) coregulate anumber of genes in mammalian cells. Ezh2 (Enhancer of zeste homolog 2)protein is the enzymatic component of the PRC2, which represses geneexpression by methylating lysine 27 of histone H3 (H3K27) (Qi et al.,Proc Natl Acad Sci USA. 2012 Dec. 26; 109(52):21360-5). Modulation ofEzh2 histone methyltransferase provides another pathway for increasingAtoh1 expression.

Modulation of the Epigenetic Status of Atoh1

Generally speaking, epigenetic regulation of gene expression involvestwo major mechanisms, DNA methylation (i.e., at the CpG islands of genepromoters) and chromatin remodeling. The transition betweentranscriptionally silent heterochromatin to active euchromatin isthought to be controlled in part by proteins that add or removemodifications, e.g., acetylation or methylation, to histones, e.g.,histone acetyltransferases (HATs); histone methyltransferases (e.g.,protein arginine methyltransferases (PRMTs) and histone lysinemethyltransferases (HKMTs)); histone deacetylases (HDACs) and histonelysine demethylases (KDMs, the lysine-specific demethylases (LSD), andthe Jumonji C (JmjC) families. See, e.g. Rotili and Mai, Genes & Cancer,2011, 2(6):663-679.

The present disclosure provides that the epigenetic status of cochleargenes can be modulated to promote sensory epithelial cell proliferationand hair cell differentiation. Accordingly, the present disclosureprovides compositions and methods for modulating cell number and Atoh1levels in target cells to promote differentiation of the target cellstowards or to a hair cell. These modifications can increase the activityof the Notch and Wnt pathways leading to proliferation of supportingcells. The proliferation can be due to increased expression of Wntdownstream targets such as cyclinD1, c-myc and n-myc. Epigeneticmodification of cochlear genes can be achieved, e.g., by effecting anincrease in H3K4 and a decrease in H3K9 and H3K27 methylation.

Thus, the methods described herein include the administration ofcompounds that modulate the epigenetic status of Atoh1. Such compoundsinclude histone deacetylase (HDAC) inhibitors, DNA methyltransferases(DNMT) inihibitors, and Histone Methyl Transferase (HMT) inhibitors.

A number of HDAC inhibitors are known in the art, including but notlimited to: Sodium Butyrate, Trichostatin A, hydroxamic acids, cyclictetrapeptides, trapoxin B, depsipeptides, benzamides, electrophilicketones, aliphatic acid compounds, pyroxamide, valproic acid,phenylbutyrate, valproic acid, hydroxamic acids, romidepsin; CI-994(N-acetyldinaline, also tacedinaline); vorinostat (SAHA), belinostat(PXD101), LAQ824, panobinostat (LBH589), Entinostat (SNDX-275; formerlyMS-275), EVP-0334, SRT501, CUDC-101, JNJ-26481585, PCI24781, Givinostat(ITF2357), and mocetinostat (MGCD0103).

A number of DNMT inhibitors are known in the art, including azacytidine,decitabine, Zebularine(1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one), procainamide,procaine, (−)-epigallocatechin-3-gallate, MG98, hydralazine, RG108, andChlorogenic acid. See also Gros et al., Biochimie. 2012 November;94(11):2280-96.

A number of EZH2/HMT inhibitors are known in the art, including but notlimited to: EPZ005687; E7438; EI1 (Qi et al., 2012, supra); EPZ-6438;GSK343; BIX-01294, UNC0638, BRD4770, EPZ004777, AZ505 and PDB 4e47, andthose described in Garapaty-Rao et al., Chem. Biol. 20(11):1329-1339(2013); Ceccaldi et al., ACS Chem Biol. 2013 Mar. 15; 8(3):543-8; US20130303555; and WO2012/005805; see, e.g., Wagner and Jung, NatureBiotechnology 30:622-623 (2012), and Yao et al., J Am Chem Soc. 2011Oct. 26; 133(42):16746-9. In some embodiments, inhibitors that act onthe G9A H3K9 methyltransferase, are used, e.g., BIX-01294 or BRD4770.

Histone Lysine Demethylase (KDM) inhibitors can also be used, e.g.,tranylcypromine ((trans-2-phenylcyclopropyl-1-amine, trans-2-PCPA)) andanalogs thereof, e.g., with substitutions at the benzene ring, e.g.,tranylcypromine 7, trans-2-PCPA analogue 28(trans-2-pentafluorophenylcyclopropylamine, 2-PFPA), and trans-2-PCPAanalogues carrying 4-bromo, 4-methoxy, and 4-trifluoromethoxysubstitutions at the benzene ring (see, e.g., Gooden et al., Bioorg MedChem Lett. 2008; 18(10):3047-51; Binda et al., J Am Chem Soc. 2010;132(19):6827-33; Mimasu et al., Biochemistry. 2010; 49(30):6494-503;Benelkebir et al., Bioorg Med Chem. 2011; 19(12):3709-16; and Mimasu etal. Biochem Biophys Res Commun. 2008; 366(1):15-22) or other inhibitors,e.g., 2,4-pyridinedicarboxylic acid (2,4-PDCA) (see, e.g., Kristensen etal., FEBS J. 2012 June; 279(11):1905-14), and inhibitors of jumonji C(jmjC)-containing KDMs, e.g., 5-Carboxy-8-hydroxyquinoline (IOX1) andn-octyl ester thereof, as described in Schiller et al., ChemMedChem.2014 March; 9(3):566-71, or other inhibitors, as described in Spannhoffet al., ChemMedChem. 2009; 4(10):1568-82; Varier and Timmers, BiochimBiophys Acta. 2011; 1815(1):75-89; Luo et al., J Am Chem Soc. Jun. 22,2011; 133(24): 9451-9456; and Rotili et al., J Med Chem. 2014 Jan. 9;57(1):42-55. Compositions can include one, two, or more compounds oragents that modulate the epigenetic status of cochlear genes,independently or otherwise. In addition, compositions can be adapted aspharmaceutical formulations.

Wnt/Beta-catenin, Hic1, and Lgr5

Wnt has been implicated in epigenetic control of gene expression,influencing the transcription of specific genes by altering theepigenetic structure of DNA. Enhanced Wnt/β-catenin signaling can driveLgr5-positive cells present in the inner ear to act as hair cellprogenitors (Shi et al., Proc Natl Acad Sci USA. Aug. 20, 2013; 110(34):13851-13856). Beta-catenin acts by binding to members of the Tcf/Leffamily for DNA binding (Tcf4 is active in the cochlea), which, likeSox2, also change the structure of DNA through an HMG domain (Brantjeset al., Biol Chem 383:255-261, 2002).

Hypermethylated in cancer 1 (Hic1), on chromosome 17p13.3, is frequentlyhypermethylated or deleted in human tumors. Evidence indicates that itis a tumor suppressor, as stable transfection in various cancer celllines results in a significant decrease in their clonogenic survival. Inaddition to effects on direct binding to DNA promoter and enhancerregions, Hic1 sequesters Tcf-4 and beta-catenin, thereby preventing themfrom associating with the TCF-binding elements of the Wnt-responsivegenes and reducing their effects on transcription. Hic1 overexpressionsuppresses TCF-mediated transcription. Hic1 RNAi has been shown toincrease the basal expression and Wnt-responsiveness of the Axin2 gene,which is another Wnt target. See, e.g., Valenta et al., EMBO Journal(2006) 25, 2326-2337. The sequence 5′-(C)/(G)NG(C)/(G)GGGCA(C)/(A) CC-3′(SEQ ID NO:5) has been identified as optimal binding site sequence forHic1, see Pinte et al., J Biol Chem. 2004 Sep. 10; 279(37):38313-24.

As shown in Example 11, reducing Hic1 activity, particularly incombination with increased beta-catenin, results in a dramatic increasein Atoh1 expression. Thus decreasing HIC1 activity provides anotherpathway for increasing Atoh1 expression.

Lgr5 is a co-receptor for Wnt signaling. Drugs that act as agonists orantagonists for the ligands of Lgr5, e.g., the R-spondins, are alsouseful in the methods described herein. See, e.g., de Lau et al., GenomeBiol. 2012;13(3):242; US 20070059829; US 20050130145; U.S. Pat. No.7,541,431.

Downstream targets of the Wnt pathway, such as c-myc and n-myc, aremediators of many of the effects of Wnt signaling and are also usefultargets for cochlear cell regeneration. Activators of c- and n-myc orWnt agonists that increase the level of these proteins can therefore beused in the present methods as well, e.g., Wnt agonists (e.g., asdescribed in Angew. Chem. Int Ed. 44, 1987-90, 2005 and WO2010/060088),and inhibitors of Wnt inhibitors, e.g., interfering RNA (siRNA, shRNA)directed against DIckopf, WIF-1, shisa, kremen, SOST. sFRP, or axin.

Inhibition of Hic1 can be achieved by administration of an inhibitorynucleic acid that specifically reduces expression of Hic1, e.g., via RNAinterference using an siRNA, shRNA, or antisense oligonucleotide. Thesequence of human Hic1 is available in GenBank at Accession no.NM_006497.3 (hypermethylated in cancer 1 protein isoform 1) orNM_001098202.1 (hypermethylated in cancer 1 protein isoform 2, thelonger isoform), and methods for making and delivering inhibitorynucleic acids that target a specific sequence are known in the art, see,e.g., Ramachandran and Ignacimuthu, Appl Biochem Biotechnol. 2013 March;169(6):1774-89; Li et al., J Control Release. 2013 Dec. 10;172(2):589-600; Lochmatter and Mullis, Horm Res Paediatr. 2011;75(1):63-9; Higuchi et al., BioDrugs. 2010 June; 24(3):195-205; and Minget al., Expert Opin Drug Deliv. 2011 April; 8(4):435-49. The inhibitorynucleic acids can be DNA, RNA, DNA/RNA hybrids, or modified, e.g., asdescribed in WO2012087983.

Pharmaceutical Formulations

The methods described herein include the manufacture and use ofpharmaceutical compositions that include compounds identified herein,e.g., histone deacetylase (HDAC) inhibitors and/or DNMT inhibitorsand/or Ezh2 Histone Methyl Transferase (HMT) inhibitors, as activeingredients. Also included are the pharmaceutical compositionsthemselves.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions, e.g., dexamethasone; prednisone; gentamicin;brain-derived neurotrophic factor (BDNF); recombinant human insulin-likegrowth factor 1 (rhIGF-1), FGF (e.g., FGF2), and/or R-spondin;combinations of the active compounds disclosed herein can also be madeand used. The present pharmaceutical compositions are formulated to becompatible with the intended route of administration.

In some embodiments, the compositions are delivered systemically, e.g.,by parenteral, e.g., intravenous, intradermal, or subcutaneousadministration.

In some embodiments, the compositions are administered by application tothe round window membrane, e.g., application of a liquid or gelformulation to the round window membrane. Application to the roundwindow membrane can be accomplished using methods known in the art,e.g., intra-tympanic injection of a liquid or gel formulation or bydirect delivery into the inner ear fluids, e.g., using a microfluidicdevice.

In some embodiments, the compositions are delivered via a pump, e.g., amini-osmotic pump, see, e.g., Takemura et al., Hear Res. 2004 October;196 (1-2):58-68, or a catheter, see, e.g., Charabi et al., ActaOtolaryngol Suppl. 2000; 543:108-10.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, NY). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems. Liposomalsuspensions (including liposomes targeted to selected cells withmonoclonal antibodies to cellular antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. Nanoparticles, e.g., poly lactic/glycolic acid(PLGA) nanoparticles (see Tamura et al., Laryngoscope. 2005 November;115(11):2000-5; Ge et al., Otolaryngol Head Neck Surg. 2007 October;137(4):619-23; Hone et al., Laryngoscope. 2010 February; 120(2):377-83;Sakamoto et al., Acta Otolaryngol Suppl. 2010 November; (563):101-4) canalso be used.

In some embodiments, the carrier comprises a polymer, e.g., a hydrogel,that increases retention of the compound on the round window andprovides local and sustained release of the active ingredient. Suchpolymers and hydrogels are known in the art, see, e.g., Paulson et al.,Laryngoscope. 2008 April; 118(4):706-11 (describing achitosan-glycerophosphate (CGP)-hydrogel based drug delivery system);other carriers can include thermo-reversible triblock copolymerpoloxamer 407 (see, e.g., Wang et al., Audiol Neurootol. 2009;14(6):393-401. Epub 2009 Nov. 16, and Wang et al., Laryngoscope. 2011February; 121(2):385-91); poloxamer-based hydrogels such as the one usedin OTO-104 (see, e.g., GB2459910; Wang et al., Audiol Neurotol2009;14:393-401; and Piu et al., Otol Neurotol. 2011 January;32(1):171-9); Pluronic F-127 (see, e.g., Escobar-Chavez et al., J PharmPharm Sci. 2006;9(3):339-5); Pluronic F68, F88, or F108;polyoxyethylene-polyoxypropylene triblock copolymer (e.g., a polymercomposed of polyoxypropylene and polyoxyethylene, of general formulaE106 P70 E106; see GB2459910, US20110319377 and US20100273864); MPEG-PCLdiblock copolymers (Hyun et al., Biomacromolecules. 2007 April;8(4):1093-100. Epub 2007 Feb. 28); hyaluronic acid hydrogels (Borden etal., Audiol Neurootol. 2011;16(1):1-11); gelfoam cubes (see, e.g.,Havenith et al., Hearing Research, February 2011; 272 (1-2):168-177);and gelatin hydrogels (see, e.g., Inaoka et al., Acta Otolaryngol. 2009April; 129(4):453-7); other biodegradable, biocompatible polymers can beused, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Tunable self-assemblinghydrogels made from natural amino acids L and D can also be used, e.g.,as described in Hauser et al e.g. Ac-LD6-COOH (L) e.g. Biotechnol Adv.2012 May-June; 30(3):593-603. Such formulations can be prepared usingstandard techniques, or obtained commercially, e.g., from AlzaCorporation and Nova Pharmaceuticals, Inc.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

The compounds and methods described herein are appropriate for thetreatment of mammalian (e.g., human) subjects who have or are at risk ofdeveloping hearing disorders resulting from cochlear hair cell loss,preferably post-neonatal (e.g., child, adolescent or adult, e.g., abovethe age of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 years) subjects.The methods described herein can be used to treat cochlear hair cellloss and any disorder that arises as a consequence of hair cell loss inthe ear, such as hearing impairments or deafness. These subjects canreceive treatment with an agent described herein. The approach may beoptimal for treatment of acute hearing loss shortly after the damage hasoccurred, and may be less effective after longer time periods when Notchsignaling has returned to its baseline level in the adult.

In some instances, methods include selecting a subject. Subjectssuitable for treatment include those at risk of hair cell loss or withhair cell loss and/or those at risk of sensorineural hearing loss orwith sensorineural hearing loss. Any subject experiencing or at risk fordeveloping hearing loss is a candidate for the treatment methodsdescribed herein. A human subject having or at risk for developing ahearing loss can hear less well than the average human being, or lesswell than a human before experiencing the hearing loss. For example,hearing can be diminished by at least 5, 10, 30, 50% or more.

The subject can have hearing loss associated with cochlear hair cellloss for any reason, or as a result of any type of event. For example, asubject can be deaf or hard-of-hearing as a result of a physicalototoxic insult, e.g., a traumatic event, such as a physical trauma to astructure of the ear. In preferred embodiments, the subject can have (orbe at risk of developing) hearing loss as result of exposure to a suddenloud noise, or a prolonged exposure to loud noises. For example,prolonged or repeated exposures to concert venues, airport runways, andconstruction areas can cause inner ear damage and subsequent hearingloss; subjects who are subjected to high levels of environmental noise,e.g., in the home or workplace, can be treated using the methodsdescribed herein. A subject can have a hearing disorder that resultsfrom aging, e.g., presbycusis, which is generally associated with normalaging processes; see, e.g., Huang, Minn Med. 90(10):48-50 (2007) andFrisina, Annals of the New York Academy of Sciences, 1170: 708-717(2009), and can occur in subjects as young as 18, but is generally moremarked in older subjects, e.g., subjects over age 40, 45, 50, 55, 60,65, 70, 75, 80, 85, or 90. A subject can have tinnitus (characterized byringing in the ears) due to loss of hair cells. A subject can experiencea chemical ototoxic insult, wherein ototoxins include therapeutic drugsincluding antineoplastic agents, salicylates, quinines, andaminoglycoside antibiotics, e.g., as described further below,contaminants in foods or medicinals, and environmental or industrialpollutants.

In some embodiments, the methods include administering to the subject acompound described herein within one, two, three, four, five, six, orseven days, or one, two, three, four, five, or six weeks of exposure toan ototoxic insult, e.g., a physical (noise, trauma) or chemical(ototoxin) insult that results in or could result in a loss of haircells, and causes an increase in Notch signaling in the subject.

In some embodiments, a subject suitable for the treatment using thecompounds and methods featured in the invention can include a subjecthaving a vestibular dysfunction, including bilateral and unilateralvestibular dysfunction; the methods include administering atherapeutically effective amount of an agent described herein, e.g., bysystemic administration or administration via the endolymphatic sac(ES). Vestibular dysfunction is an inner ear dysfunction characterizedby symptoms that include dizziness, imbalance, vertigo, nausea, andfuzzy vision and may be accompanied by hearing problems, fatigue andchanges in cognitive functioning. Vestibular dysfunctions that can betreated by the methods described herein can be the result of a geneticor congenital defect; an infection, such as a viral or bacterialinfection; or an injury, such as a traumatic or nontraumatic injury,that results in a loss of vestibular hair cells. In some embodiments,balance disorders or Meniere's disease (idiopathic endolymphatichydrops) may be treated by the methods described herein. Vestibulardysfunction is most commonly tested by measuring individual symptoms ofthe disorder (e.g., vertigo, nausea, and fuzzy vision).

Alternatively or in addition, the compounds and methods featured in theinvention can be used prophylactically, such as to prevent, reduce ordelay progression of hearing loss, deafness, or other auditory disordersassociated with loss of hair cells. For example, a compositioncontaining one or more compounds can be administered with (e.g., before,after or concurrently with) an ototoxic therapy, i.e., a therapeuticthat has a risk of hair cell toxicity and thus a risk of causing ahearing disorder. Ototoxic drugs include the antibiotics neomycin,kanamycin, amikacin, viomycin, gentamycin, tobramycin, erythromycin,vancomycin, and streptomycin; chemotherapeutics such as cisplatin;nonsteroidal anti-inflammatory drugs (NSAIDs) such as choline magnesiumtrisalicylate, diclofenac, diflunisal, fenoprofen, flurbiprofen,ibuprofen, indomethacin, ketoprofen, meclofenamate, nabumetone,naproxen, oxaprozin, phenylbutazone, piroxicam, salsalate, sulindac, andtolmetin; diuretics; salicylates such as aspirin; and certain malariatreatments such as quinine and chloroquine. For example, a subjectundergoing chemotherapy can be treated using the compounds and methodsdescribed herein. The chemotherapeutic agent cisplatin, for example, isknown to cause hearing loss. Therefore, a composition containing one ormore compounds can be administered with cisplatin therapy (e.g., before,after or concurrently with) to prevent or lessen the severity of thecisplatin side effect. Such a composition can be administered before,after and/or simultaneously with the second therapeutic agent. The twoagents may be administered by different routes of administration.

In general, the compounds and methods described herein can be used togenerate hair cell growth in the ear and/or to increase the number ofhair cells in the ear (e.g., in the inner, middle, and/or outer ear).For example, the number of hair cells in the ear can be increased about2-, 3-, 4-, 6-, 8-, or 10-fold, or more, as compared to the number ofhair cells before treatment. This new hair cell growth can effectivelyrestore or establish at least a partial improvement in the subject'sability to hear. For example, administration of an agent can improvehearing loss by about 5, 10, 15, 20, 40, 60, 80, 100% or more.

In some instances, compositions can be administered to a subject, e.g.,a subject identified as being in need of treatment, using a systemicroute of administration. Systemic routes of administration can include,but are not limited to, parenteral routes of administration, e.g.,intravenous injection, intramuscular injection, and intraperitonealinjection; enteral routes of administration e.g., administration by theoral route, lozenges, compressed tablets, pills, tablets, capsules,drops (e.g., ear drops), syrups, suspensions and emulsions; transdermalroutes of administration; and inhalation (e.g., nasal sprays).

In some instances, compositions can be administered to a subject, e.g.,a subject identified as being in need of treatment, using a systemic orlocal route of administration. Such local routes of administrationinclude administering one or more compounds into the ear of a subjectand/or the inner ear of a subject, for example, by injection and/orusing a pump.

In some instances, compositions can be can be injected into the ear(e.g., auricular administration), such as into the luminae of thecochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani). Forexample, compositions can be administered by intratympanic injection(e.g., into the middle ear), and/or injections into the outer, middle,and/or inner ear. Such methods are routinely used in the art, forexample, for the administration of steroids and antibiotics into humanears. Injection can be, for example, through the round window of the earor through the cochlea capsule. In another exemplary mode ofadministration, compositions can be administered in situ, via a catheteror pump. A catheter or pump can, for example, direct a pharmaceuticalcomposition into the cochlea luminae or the round window of the ear.Exemplary drug delivery apparatus and methods suitable for administeringone or more compounds into an ear, e.g., a human ear, are described byMcKenna et al., (U.S. Publication No. 2006/0030837) and Jacobsen et al.,(U.S. Pat. No. 7,206,639). In some embodiments, a catheter or pump canbe positioned, e.g., in the ear (e.g., the outer, middle, and/or innerear) of a subject during a surgical procedure. In some embodiments, acatheter or pump can be positioned, e.g., in the ear (e.g., the outer,middle, and/or inner ear) of a subject without the need for a surgicalprocedure.

In some instances, compositions can be administered in combination witha mechanical device such as a cochlea implant or a hearing aid, which isworn in the outer ear. An exemplary cochlea implant that is suitable foruse with the present invention is described by Edge et al., (U.S.Publication No. 2007/0093878).

In some instances, compositions can be administered according to any ofthe Food and Drug Administration approved methods, for example, asdescribed in CDER Data Standards Manual, version number 004 (which isavailable at fda.give/cder/dsm/DRG/drg00301.htm).

In some instances, the present disclosure includes treating a subject byadministering to the subject cells produced using the compositions andmethods disclosed herein. In general, such methods can be used topromote complete or partial differentiation of a cell to or towards amature cell type of the inner ear (e.g., a hair cell) in vitro. Cellsresulting from such methods can then be transplanted or implanted into asubject in need of such treatment. Cell culture methods required topractice these methods, including methods for identifying and selectingsuitable cell types, methods for promoting complete or partialdifferentiation of selected cells, methods for identifying complete orpartially differentiated cell types, and methods for implanting completeor partially differentiated cells are described herein. Target cellssuitable for use in these methods are described above.

In some instances, methods can include administering one or morecompositions disclosed herein and cells produced using the compositionsand methods disclosed herein to a subject.

Administration of cells to a subject, whether alone or in combinationwith compounds or compositions disclosed herein, can includeadministration of undifferentiated, partially differentiated, and fullydifferentiated cells, including mixtures of undifferentiated, partiallydifferentiated, and fully differentiated cells. As disclosed herein,less than fully differentiated cells can continue to differentiate intofully differentiated cells following administration to the subject.

Where appropriate, following treatment, the subject can be tested for animprovement in hearing or in other symptoms related to inner eardisorders. Methods for measuring hearing are well-known and include puretone audiometry, air conduction, and bone conduction tests. These examsmeasure the limits of loudness (intensity) and pitch (frequency) that asubject can hear. Hearing tests in humans include behavioral observationaudiometry (for infants to seven months), visual reinforcementorientation audiometry (for children 7 months to 3 years); playaudiometry for children older than 3 years; and standard audiometrictests for older children and adults, e.g., whispered speech, pure toneaudiometry; tuning fork tests; brain stem auditory evoked response(BAER) testing or auditory brain stem evoked potential (ABEP) testing.Oto-acoustic emission testing can be used to test the functioning of thecochlear hair cells, and electro-cochleography provides informationabout the functioning of the cochlea and the first part of the nervepathway to the brain. In some embodiments, treatment can be continuedwith or without modification or can be stopped.

Dosage

An “effective amount” is an amount sufficient to effect beneficial ordesired therapeutic effect. This amount can be the same or differentfrom a prophylactically effective amount, which is an amount necessaryto prevent onset of disease or disease symptoms. An effective amount canbe administered in one or more administrations, applications or dosages.A therapeutically effective amount of a therapeutic compound (i.e., aneffective dosage) depends on the therapeutic compounds selected. Thecompositions can be administered one from one or more times per day toone or more times per week; including once every other day. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of the therapeutic compounds described herein caninclude a single treatment or a series of treatments. In someembodiments, e.g., in subjects exposed to prolonged or repeatedexposures to noise, e.g., normal noises such as are associated withactivities of daily life (such as lawnmowers, trucks, motorcycles,airplanes, music (e.g., from personal listening devices), sportingevents, etc.), or loud noises, e.g., at concert venues, airports, andconstruction areas, that can cause inner ear damage and subsequenthearing loss; e.g., subjects who are subjected to high levels ofenvironmental noise, e.g., in the home or workplace, can be treated withrepeated, e.g., periodic, doses of the pharmaceutical compositions,e.g., to prevent (reduce the risk of) or delay progression or hearingloss.

Dosage, toxicity and therapeutic efficacy of the therapeutic compoundscan be determined by standard pharmaceutical procedures, e.g., in cellcultures or experimental animals, e.g., for determining the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compounds that exhibit hightherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. For example,samples of the perilymph or endolymph can be obtained to evaluatepharmacokinetics and approximate an effective dosage, e.g., in animalmodels, e.g., after administration to the round window. The dosage ofsuch compounds lies preferably within a range of concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated from cellculture assays, and/or a dose may be formulated in animal models;alternatively, for those compounds that have been previously used inhumans, clinically desirable concentrations can be used as a startingpoint. Such information can be used to more accurately determine usefuldoses in humans.

Kits

The compositions and/or cells disclosed herein can be provided in a kit.For example, kits can include (a) one or more compounds, such as in acomposition that includes the compound, (b) cells that have been inducedto differentiate, (c) informational material, and any combination of(a)-(c). The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or to the use of the agent for the methods described herein. Forexample, the informational material relates to the use of the compoundto treat a subject who has, or who is at risk for developing, a auditoryhair cell loss hearing. The kits can also include paraphernalia foradministering one or more compounds to a cell (in culture or in vivo)and/or for administering a cell to a patient, and any combination of themethods described herein.

In one embodiment, the informational material can include instructionsfor administering the pharmaceutical composition and/or cell(s) in asuitable manner to treat a human, e.g., in a suitable dose, dosage form,or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). In another embodiment, theinformational material can include instructions to administer thepharmaceutical composition to a suitable subject, e.g., a human, e.g., ahuman having, or at risk for developing, auditory hair cell loss.

The informational material of the kits is not limited in its form. Inmany cases, the informational material (e.g., instructions) is providedin printed matter, such as in a printed text, drawing, and/orphotograph, such as a label or printed sheet. However, the informationalmaterial can also be provided in other formats, such as Braille,computer readable material, video recording, or audio recording. Ofcourse, the informational material can also be provided in anycombination of formats.

In addition to the compound, the composition of the kit can includeother ingredients, such as a solvent or buffer, a stabilizer, apreservative, a fragrance or other cosmetic ingredient, and/or a secondagent for treating a condition or disorder described herein.Alternatively, the other ingredients can be included in the kit, but indifferent compositions or containers than the compound. In suchembodiments, the kit can include instructions for admixing the agent andthe other ingredients, or for using one or more compounds together withthe other ingredients.

The kit can include one or more containers for the pharmaceuticalcomposition. In some embodiments, the kit contains separate containers,dividers or compartments for the composition and informational material.For example, the composition can be contained in a bottle (e.g., adropper bottle, such as for administering drops into the ear), vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, thecomposition is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label. In someembodiments, the kit includes a plurality (e.g., a pack) of individualcontainers, each containing one or more unit dosage forms (e.g., adosage form described herein) of the pharmaceutical composition. Forexample, the kit can include a plurality of syringes, ampoules, foilpackets, or blister packs, each containing a single unit dose of thepharmaceutical composition. The containers of the kits can be air tightand/or waterproof, and the containers can be labeled for a particularuse. For example, a container can be labeled for use to treat a hearingdisorder.

As noted above, the kits optionally include a device suitable foradministration of the composition (e.g., a syringe, pipette, forceps,dropper (e.g., ear dropper), swab (e.g., a cotton swab or wooden swab),or any such delivery device).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: Expression of Sox2 and Pax2 in Stem Cells Differentiatinginto Hair Cells

As discussed above, Sox2 is an HMG domain transcription factor whosebinding to DNA is enhanced and tissue-specific functions are conferredby partner interactions with a number of other transcription factors(Ambrosetti et al., Mol. Cell. Biol., 17:6321-6329 (1997); Kamachi etal., Genes Dev., 15:1272-1286 (2001); Kondoh et al., Int. J. Dev. Biol.,48:819-827 (2004)), and Pax2 is a homeodomain transcription factor thatbinds to DNA through its paired domain. Both Pax2 and Sox2 are expressedduring development of the otocyst and are important for the developmentof the cochlea during tissue morphogenesis. Pax2 and Sox2 are bothexpressed in inner ear stem cells (Martinez-Monedero et al., Dev.Neurobiol., 68:669-684 (2008)). Their level of expression decreases wheninner ear stem cells are transferred from a proliferative self-renewingculture (as floating neurospheres in growth factors) to adifferentiating culture (as adherent cells in the absence of growthfactors) (Martinez-Monedero et al., supra). The relationship betweenSox2 and Pax 2 and the role of such relationship in the inner ear wasinvestigated as described in the following examples.

Cellular expression profiles of Sox2 and Pax2 in stem cellsdifferentiating into hair cells were reviewed usingimmunohistochemistry. Permeabilization and blocking was performed withblocking solution (0.3% Triton X-100, 15% heat inactivated goat ordonkey serum in PBS) for 1 h. Diluted primary antibody (0.1% TritonX-100, and 10% heat inactivated goat or donkey serum in PBS) was appliedovernight at 4° C. Antibody dilutions were: 1:100 for monoclonal mouseantibody against myosin VIIa (Developmental Studies Hybridoma Bank),1:100 for polyclonal rabbit antibody against Pax2 (Covance), and 1:300for polyclonal goat antibody against Sox2 (Santa Cruz). Secondaryantibodies (Alexafluor 488, 568, and 647-conjugated; Invitrogen) wereused for detection of primary antibodies. Nuclei were visualized with4,6-diamidino-2-phenylindole (Vector Laboratories). Staining wasanalyzed with epifluorescence microscopy (Axioskop2 Mot Axiocam, Zeiss)and confocal microscopy (TCD, Leica).

At day 3, differentiating cells exhibit patches of Pax2 and Sox2expression with little overlap. At day 7, all cells that co-labelled forSox2 and Pax2 showed robust expression of hair cell markers, Atoh1 ormyosin VIIa. In a few cells, myosin VIIa positive cells were positivefor Sox2 or Pax2, but not both. By day 10, most hair cell-like cells nolonger expressed Pax2 or Sox2. About 30% of Atoh-1-positive cellsco-expressed Pax2 and Sox2.

Example 2: Pax2 and Sox2 Co-Expression in Embryonic Hair CellProgenitors

Expression of Pax2 and Sox2 was analyzed in vivo during development ofmouse cochlea.

Embryos were collected at E12.5, 14.5, 15.5 and 17.5 (withidentification of a positive plug in the morning counted as E0.5) andfixed in 4% paraformaldehyde for 4 hours at 4° C. After dehydration withsucrose (5% and 30%), embryos were embedded in OCT and kept at −80° C.For immunohistochemistry, tissue was cut (12 μm) and then stained asdescribed in Example 1.

Expression of Pax2 and Sox2 was observed in the cochlea and vestibularsystem at the E12.5 time point. Furthermore, the observed expressionpattern was segregated, with Pax2 seen on the abneural side of thecochlea and Sox2 on the neural side. A similar pattern was seen in thevestibular organs. Little overlap was observed in the ventromedialregion and the future sensory epithelium.

At E14.5 in the cochlear duct, around the time of hair cell formation,only cells in the upper layer of the sensory epithelium, from which haircells arise, showed overlapping expression for Pax2 and Sox2. At E15.5,Pax2-Sox2 co-expression was only seen in the area of maturing haircells. At E17.5, hair cells continued to co-express Pax2 and Sox2 andshowed strong myosin VIIa expression. These results in cochlear stemcells and in balance and hearing organs of the developing inner earsuggested that co-expression of these transcription factors occurs priorto the development of hair cells.

At P0, myosin VIIa-positive hair cells had lost most Sox2 expression,and Pax2 expression was lost in most inner hair cells. Developingsupporting cells at all stages only stained for Sox2 and did notco-label for Pax2. In the vestibular system at E15.5, only developingmyosin VIIa-positive utricular and saccular hair cells showedco-labeling for Pax2 and Sox2. Despite broad expression in thedeveloping otocyst, Pax2 and Sox2 were expressed in different regions ofthe cochlea and did not overlap except in cells that were destined tobecome hair cells.

Example 3: Interaction Between Pax2 and Sox2 and the Atoh1 3′ Enhancer

The above-reported co-localization of Pax2 and Sox2 in hair cells duringdevelopment and in new hair cells from inner ear stem cells suggested animportant role for the transcription factors in the generation of haircells. Since both transcription factors bind to DNA and act astranscriptional activators, a search was performed to identify consensussequences, within the highly conserved murine and human Atoh1 3′enhancers, that may be sufficient to confer Pax2/Sox2-dependentheterologous Atoh1 expression in transgenic mice. This search identifieda highly conserved site containing elements of adjacent Sox2 and Pax2consensus sequences (see FIGS. 1A-B (boxes indicate additional bindingsites for Pax2 and Sox2)). Consensus sites were separated by 2 bases.

To confirm that the above-identified putative binding sites are bound bySox2 or Pax2, chromatin immunoprecipitation (ChIP) was performed in OC-1cells, an inner ear cell line established from embryonic immorto-mousecochlea that endogenously expresses many of the markers of outer haircells, and shows robust expression of Atoh1 (Kalinec et al., Genes Dev.,15:1272-1286 (1999)). Endogenous levels of Pax2 and Sox2 levels are lowin OC-1 cells. To address this, Pax2 and Sox2 cDNA was transfected.

For ChIP, 10 cm² culture dishes with OC-1 cells were seeded andtransfected at 30% confluency using Lipofectamine (3 μl/μg DNA/ml) andPax2 or Sox2 cDNA (0.5 μg/ml each) in Opti-MEM. After 48 h, cells wereharvested and processed for ChIP. Chromatin was precipitated with eithergoat anti-Sox2 antibody (3 μg) or rabbit anti-Pax2 antibody (3 μg) orcontrol non-immune goat serum or normal rabbit IgG (3 μg). Target DNAwas amplified by PCR for Atoh1 regulatory regions using the primersshown in Table 1

TABLE 1 Target (binding site) Sense Antisense    4-193AAGGTCCGGCAATGAAGTTT AAAGGAACCAGTCAGCATGG (SEQ ID NO: 6) (SEQ ID NO: 7) 153-375 CCATATGCCAGACCACTCCT GCGGTGTCCCAAAGAACTAA (SEQ ID NO: 8)(SEQ ID NO: 9)  352-512 CGGGTTAGTTCTTTGGGACA GCTCCCCGTGAAATCAAATA(SEQ ID NO: 10) (SEQ ID NO: 11)  446-637 GGTTTTGGCTCACCACACTTCTCTGGTCTCCTGCTGGTTC (SEQ ID NO: 12) (SEQ ID NO: 13)  570-815CGAATGGCACATCTACCAGA CGCGATCTTCACCTCTCAGT (SEQ ID NO: 14)(SEQ ID NO: 15)  808-968 CGGGTTAGTTCTTTGGGACA GCTCCCCGTGAAATCAAATA(SEQ ID NO: 16) (SEQ ID NO: 17)  936-1108 CTAGTGTCTCCCCAGGCAAGAAACTACCCCCACGCTTCTT (SEQ ID NO: 18) (SEQ ID NO: 19) 1089-1332AAGAAGCGTGGGGGTAGTTT AGCAAGGCTGTCTACGAGGA (SEQ ID NO: 20)(SEQ ID NO: 21) 1311-1493 CCTCCTCGTAGACAGCCTTG TGGCTTAAGCATGCTCCTTT(SEQ ID NO: 22) (SEQ ID NO: 23)

Pax2 or Sox2 antibody was used to precipitate proteins bound to the DNAof OC-1 cells and PCR was performed for the entire enhancer.

For knockdown experiments, cells were transfected with siRNA (100 nMON-TARGET plus, smart pool from Dharmacon for Pax2, Sox2, ornon-targeting siRNA), using Gene Silencer (Genlantis) for 24 h accordingto the manufacturer's instructions. In addition, 2.5 μM DAPT was addedduring differentiation culture.

As shown in FIG. 2A, both Pax2 and Sox2 bound the DNA of the Atoh1enhancer in the area of the putative binding site between bases 303-335.This indicated an active binding site for Pax2 and Sox2 on the Atoh1 3′enhancer. As shown in FIG. 2B, Pax2 and Sox2 antibodies, but not controlantibodies, also precipitated DNA in the region of the binding site inHEK cells after transfection of Pax2 and Sox2 along with the Atoh1 3′enhancer construct represented in FIG. 3A. Antibodies for both Pax2 andSox2 also precipitated a construct containing a 4×-repeat of thePax2-Sox2 binding site from OC-1 cells (FIG. 2C and FIG. 3B).

After treatment with siRNA for Sox2 or Pax2, less DNA was precipitatedwith Pax2 antibody from HEK cells transfected with the 4×-binding siteconstruct (FIG. 2D).

Example 4: Interaction Between Pax2 and Sox2

Direct protein-protein interaction between Pax2 and Sox2 was evaluatedby immunoprecipitation (IP).

For transfection, OC-1 cells or spheres were seeded in DMEM/10% FBS at40-50% confluency. Cells were transfected in optiMEM (GIBCO) with Pax2or Sox2 cDNA or both (2 ng total for OC-1 cells, 20 ng total for spheresdue to low transfection efficiency) and empty vector as a control. DNAwas normalized to 2 ng or 20 ng total with empty vector. Transfectionagent was Lipofectamine (3 μl/μg DNA/ml, Invitrogen). Transfection wasstopped after 48 h, cells were harvested on ice and RNA was extractedimmediately.

For immunoprecipitation, magnetic Dynabeads Protein G beads (Invitrogen)were incubated with 10 μg of either anti-Pax2 antibody or anti-Sox2antibody or control normal goat or rabbit IgG at 4° C. overnight.Antibody supernatant was removed and beads were washed according to aDynal IP Kit (Invitrogen). Beads were then incubated with cell lysate orchromatin lysate of HEK cells for 4 h at room temperature, followed bywashing; protein was eluted with elution buffer and SDS sample buffer at70° C. for 10 min. Elution product was loaded onto acrylamide gel andWestern blot analysis was performed.

As shown in FIG. 4A, Pax2 antibody (and Sox2 as a control) precipitatedSox2 protein from OC-1 cells transfected with Pax2 and Sox2, whilecontrol IgG did not. Using cellular lysates of HEK cells transfectedwith Pax2 and Sox2, Pax2 antibody co-precipitated Sox2, and Sox2antibody co-precipitated Pax2 (FIG. 4B), whereas neither Pax2 nor Sox2were precipitated in the control. These data indicated that Sox2 andPax2 interact directly. In addition, when nuclear lysate from HEK cellswas used for IP, Sox2 antibody precipitated Pax2 and Pax2 antibodyprecipitated Sox2 (FIG. 4C). Thus, Pax2 and Sox2 were present in thecomplex and bound to DNA and to each other.

Example 5: The Pax2-Sox2 Consensus Sequence is Functionally Active

Enhancer activity was assessed via luciferase reporter assay in IEC6cells. IEC6 cells were seeded onto 96-well plates 1 d beforetransfection. At 40-50% confluency, 100 ng of luciferase reporterconstruct (intact Atoh1 enhancer or 4×-binding site reporter or mutatedcontructs), 10 ng of Renilla luciferase construct and different amountsof Pax2 and Sox2 effector (from 0.1 ng-100 ng) were transfected withLipofectamine (3 μl/μg DNA/ml) and incubated with the cells for 6 h. Twoluciferase reporter constructs were transfected containing either thefull-length Atoh1 3′ enhancer or a 4×-repeat of the Pax2-Sox2 bindingsite (FIG. 3).

For RNAi, 100 nM total siRNA was transfected with GeneSilencer for 24 h.Cells were lysed after 48 h, and luciferase activity was measured usingthe Dual Luciferase Reporter Assay System (Promega) according to themanufacturer's instructions in a Wallac Victor2 1420 Multilabel Counter(Perkin-Elmer).

As shown in FIG. 5, transfection of Pax2 or Sox2 cDNA alone did not leadto significant activation of the Atoh1 3′ enhancer. However, as shown inFIG. 5, right column, and FIG. 6, combination of equal amounts of Pax2and Sox2 cDNA significantly increased activity. This increasedactivation with the single factors in the full-length enhancer could bedue to individual sites for Pax and Sox proteins that could be bound atlow affinity by Pax2 and Sox2.

As shown in FIG. 7A, addition of siRNA for Pax2 and Sox2 aftertransfection of Pax2 and Sox2 cDNA (1 ng each) decreased activation ofAtoh1. Activation of the Atoh1 enhancer was the greatest with low Pax2and Sox2 concentrations (1 ng each) and high concentrations of Sox2decreased activation of Atoh1 (FIG. 7B).

Similar experiments were conducted using a full-length Atoh1 3′ enhancer(see FIG. 8A) in a neural cell line, Neuro2a, and HEK cells. As shown inFIGS. 8B-C, slight differences were seen in the concentrations of Pax2and Sox2 needed for optimal activation, but similar for all cell lines.Only low concentrations of Pax2 and Sox2 activated the enhancer, andhigh concentrations of Sox2 abolished the effect.

Example 6: Characterization of the Pax2-Sox2 Binding Site for Atoh1Activation

Multiple mutations were introduced into the Pax2-Sox2 binding site forAtoh1 activation to further assess activity of the site.

In mutation 1, the spacing between the two binding sites was altered bythe addition of 8 bases. Mutation 2 contained mutated Pax2 and Sox2binding sites (see FIG. 3). Promoter activity was then assessed viareporter assay, conducted as described above.

As shown in FIG. 9A, both mutation 1 and 2 significant reduced promoteractivity and addition of Pax2 or Sox2 cDNA alone did not produce anyactivation in either mutated construct (FIGS. 9B and 9C). The promotercontaining mutation 1 was activated when Pax2 and Sox2 were addedtogether, but the promoter containing mutation 2 was not (FIG. 9D).

These data suggests that activation of the Atoh1 promoter requiresinteraction and binding of Pax2 or Sox2 to the Pax2 or Sox2 promoterbinding site. In addition, activation required both Pax2 or Sox2 bindingsites and maintenance of the wild-type spacing of the two sequences wascritical for the protein-protein interaction.

Example 7: Levels of Pax2 and Sox2 Impact Promoter Activity

To confirm that levels of Pax2 or Sox2 affects activation of theenhancer RNAi was performed. siRNA against Sox2 and Pax2 was introducedinto cells as described above. As shown in FIG. 10, after transfectionof Pax2, promoter activity was reduced by 50%, compared to controlsiRNA, indicating a crucial role for Pax2 in activation of the bindingsite.

Atoh1 levels were measured in vitro in OC-1 cells or spheres byquantitative RT-PCR. For RT-PCR, total RNA was extracted from OC-1 cellsor spheres treated with DAPT, FGF2, or DAPT and RNAi with the RNeasyMaxi Kit (Qiagen) according to the manufacturer's instructions. RNA wasdenatured at 65° C. for 5 min. For reverse transcription, ImProm II(Promega) was used with random hexamers. Reverse transcriptionconditions were 25° C. for 5 min followed by 42° C. for 60 min.Reactions were terminated at 70° C. for 15 min. cDNAs were mixed withPlatinum quantitative PCR Supermix ROX with UDG (Invitrogen) and primersfor Atoh1, Pax2 or Sox2 (ABI) according to the manufacturer'sinstructions. Gene expression was measured relative to 18S RNA. Sampleswere analyzed in 96 well plates in triplicate by quantitative PCR(Applied Biosystems 7900HT) using the following conditions: initialdenaturation at 95° C. for 2 min, denaturation at 95° C. for 40 s andannealing/extension at 60° C. for 35 s for 45 cycles.

As shown in FIG. 11, transfection with a combination of Pax2 and Sox2led to a strong increase in Atoh1, whereas single transfection had noeffect. Under stimulating conditions with DAPT, siRNA for Pax2 or Sox2had little effect, but combined siRNAs for Pax2 and Sox2 significantlydecreased Atoh1 as compared to control siRNA (relative ratio compared tocontrol siRNA of 0.147±0.049 SEM, p<0.001 for OC-1 cells and 0.427±0.132SEM, p<0.05 for spheres).

Example 8: Increasing Differentiation of Hair Cells from Inner Ear StemCells Using Pax2 and Sox2

To confirm that modulation of Pax2-Sox2 expression levels leads to theformation of hair cells, modulation of Pax2 and Sox2 expression wastriggered in differentiating inner ear stem cells.

For each experiment, tissue of neonatal pups with C57BL/6 (Jackson Labs)or Atoh1-nGFP background were dissected on ice in HBSS and the OC washarvested after removal of spiral ganglion neurons and stria vascularisas previously described (Parker et al., 2010).

Embryos were collected at E12.5, 14.5, 15.5 and 17.5 (withidentification of a positive plug in the morning counted as E0.5) andfixed in 4% paraformaldehyde for 4 h at 4° C. After dehydration withsucrose (5% and 30%), embryos were embedded in OCT and kept at −80° C.For immunohistochemistry, tissue was cut (12 μm) and then stained asdescribed for cultured cells.

For each experiment, cochleae of 4-6 neonatal C57BL/6 or Atoh1-nGFP pups(Lumpkin et al., Gene Expr. Patterns, 3:389-395 (2003)) that express GFPunder the control of the Atoh1 enhancer (provided by Jane E. Johnson)were dissected in HBSS and the organ of Corti (OC) was separated fromthe stria vascularis and the spiral ganglion neurons. Tissues weredissociated in trypsin (0.25%) for 13 min in PBS at 37° C. 10% FBS inDMEM-high glucose medium was used to stop the reaction. After washing,tissue was manually dissociated. Titurated cells were then passedthrough a 70 μm cell strainer (BD Labware) to remove tissue debris.Single cells were cultured in DMEM-high glucose medium and F12 (1:1)supplemented with N2 and B27 (Invitrogen), and EGF (20 ng/mL; Chemicon),bFGF (10 ng/mL; Chemicon), IGF-1 (50 ng/mL;Chemicon), and heparansulfate (50 ng/mL; Sigma). Single cells were maintained in ultra-lowcluster plates (Costar) for several days in culture to obtain spheres14. For passage, spheres of the first generation were dissociated with a25G needle and syringe (BD Labware) 6-8 times. Single cell suspensionswere cultured in fresh medium F12/DMEM (1:1) with the same growthfactors to form spheres until use at the 4th to 5th generation.

In this example, modulation of Pax2 and Sox2 expression in thedifferentiating inner ear cells included reducing Sox2 expression levelswhile increasing Pax2 expression level. Sox2 expression was modulatedusing DAPT (Jeon et al., j. Neurosci., 31:8351-8358 (2011)). Pax2expression was modulated using FGF2 (Adamska et al., Mech. Dev.,109:303-313 (2001); Mansukhani et al., J. Cell. Biol., 168:1065-1076(2005)). DAPT was used to decrease Sox2 and FGF2 to activate Pax2expression in inner ear stem cells (see FIG. 12A). 4th-5th generationspheres or OC explants (P1-P 2) were plated in 4-well plates (Greiner)on round 10 mm glass coverslips coated with poly-L-lysine (Cultrex) andattachment took place overnight in 10% FBS/DMEM-high glucose (GIBCO).Attachment was ensured with microscopic inspection and the medium waschanged to serum-free DMEM-high glucose/F12 (mixed 1:1, GIBCO) and N2and B27 (Invitrogen). Spheres were differentiated for 3, 7 or 10 d. Fortreatment, either DAPT (2.5 μM) or FGF (20 ng/ml) or control mediumcontaining only DMSO (0.1%) were applied for 7 d on spheres and 48 h onOC explants. Cells were harvested and further analyzed byimmunohistochemistry. Axiovision 4.3 was used for data acquisition andthe number of cells was quantified with Metamorph software. Cell countswere expressed as mean±standard deviation. An average of 1,000 cellswere counted for spheres or 100 μm for OC explants. Origin software wasused for statistical evaluation. Cell detection techniques are describedin the Examples above.

As shown in FIG. 12A, an increase in Atoh1-GFP or myosin VIIa positivecells was apparent after 7 d. The combination of FGF and DAPT producedthe highest number of hair cell marker-positive cells (FIG. 12A). Thenumber of Pax2 and Sox2 positive cells increased after FGF and DAPTtreatment (from 29.52%±4.52% SEM of myosin VIIa-positive cells incontrol to 37.61%±4.28% SEM in treated spheres).

To confirm that the increased differentiation was specifically caused byan effect of DAPT on Pax2 and Sox2, Pax2 and Sox2 expression wasinhibited using siRNA (as described above) under stimulating conditionswith DAPT. Hair cell number was analyzed at day 7 based on myosin VIIastaining and Atoh1-GFP expression. As shown in FIG. 16B, siRNAs for Pax2or Sox2 substantially decreased the number of hair cells, while controlsiRNA did not have an effect on the number of hair cells. As shown inFIG. 16B, the combination of siRNAs for Pax2 and Sox2 further decreasedthe number of hair cells, confirming that modulation of both Pax2 andSox2 was needed for hair cell differentiation.

Example 9: Increasing Hair Cell Differentiation in the Organ of Corti

Postnatal hair cells in the organ of Corti have lost the ability toregenerate and are Pax2 and Sox2 negative. Previous data have shownthat, when treated with DAPT, new hair cells form in the apical regionof organ of Corti explants in culture (Doetzlhofer et al., Dev. Cell,16:58-69 (2009); Yamamoto et al., J. Mol. Med., 84:37-45 (2006)). Theimpact of Pax2-Sox2 modulation of hair cell differentiation in the organof Corti was assessed by contacting isolated organ of Corti with DAPTand/or FGF.

Samples were obtained and detected using methods described above. Organof Corti explants (P1-P2) from Atoh1-GFP mice were contacted with DAPT,FGF2, or a combination of both for 48 h. Hair cell number was thenassessed.

As shown in FIGS. 13A-B, no new hair cells were observed in the control,untreated, organ of Corti and any hair cells were Atoh1-GFP positive andPax2 and Sox2 negative. In contrast, in both FGF2 and DAPT treatedexplants, additional outer hair cells were seen in the apex.Furthermore, the combination was the most effective at producing newhair cells (see FIG. 13A). In addition, the new hair cells co-expressedPax2 and Sox2 (see FIG. 13B). Expression of Pax2 and Sox2 was lost at 72h in culture. These data suggest that Pax2 was transiently upregulatedin supporting cells, leading to Pax2-Sox2 activation of Atoh1, whichresulted in direct transdifferentiation of supporting cells.

To confirm that DAPT had a direct effect on Pax2 and Sox2 regulation,postnatal organ of Corti explants were treated with siRNA for Pax2 andSox2 under stimulating conditions with DAPT. Increased numbers of haircells were observed after treatment with DAPT and control siRNA. Incontrast, new hair cell formation was strongly inhibited by siRNAi forPax2 or Sox2. These results underline the importance of Pax2 and Sox2for activation of Atoh1 and regeneration of hair cells in the organ ofCorti.

Example 10: Epigenetic Regulation of Atoh1

As described above in Examples 1-9, Sox2, a high-mobility grouptranscription factor, and Pax2, a paired homeodomain transcriptionfactor, when co-expressed in inner ear stem cells always led to haircell differentiation. In the developing otocyst, expression of Pax2 andSox2 overlapped at the site of hair cell formation.

The finding that Sox2 bound to the Atoh1 enhancer led to the possibilityof epigenetic regulation of Atoh1. Epigenetic regulation is suggested bythe known role of Sox2 and Wnt signaling in stem cells (Kopp et al.,2008; Marson et al., 2008; Kim et al., 2009; Engelen et al., 2011). Sox2directly alters DNA structure by binding to DNA through its HMG domainthat inserts into the minor groove and bends the double helix (Engelenet al., 2011).

The effects of Sox2 are mediated by CHD7 in several organs, includingthe ear, by regulation of epigenetic signaling (Hurd et al., 2010;Engelen et al., 2011). The enzymatoc activity of CHD7 which binds toH3K4me sites, modifies chromatin to change the interaction of DNA withhistones (Engelen et al., 2011). Its deficiency results in Chargesyndrome, which has several severe manifestations, including deafness.The effects of Pax2 are mediated by its association with DNA bindingprotein, PTIP (Patel et al., 2007). PTIP has a known role in histonemodifications through a direct association with H3 methyltransferase,KMT2a. PTIP together with Pax2 lead to a pattern of methylationcharacteristic of actively transcribed genes, an increase in H3K4 and adecrease in H3K9 and H3K27 methylation (Patel et al., 2007; Kim et al.,2009).

Cochlear expression of PTIP was assessed. Expression of CHD7 had beendescribed, but PTIP was not known to be present in the cochlea. The P1organ of Corti, where Sox2 and Pax2 pathways are active, showed strongexpression of PTIP.

Changes in relative levels of H3K4Me3, H3K27Me3 and EZH2 at the Sox2 andAtoh1 loci were evaluated in undifferentiated and differentiated haircell progenitor cell line, VOT-E36. Chromatin immunoprecipitation (ChIP)was performed using ActiveMotif© High Sensitivity protocol withantibodies to H3K4Me3, H3K27Me3, EZH2 and IgG (control). qPCR toregulatory regions of Atoh1 and Sox2 was performed using publishedprimers (Bardot et al., 2013). Fold enrichment relative to IgG ispresented in FIGS. 14A-B. Sox2 is a key stem cells gene also found insupporting cells in the inner ear. Atoh1 is a known Sox2 target, andSox2 upregulates Atoh1 expression. Activating H3K4Me3 and inhibitoryH3K27Me3 chromatin marks, along with EZH2 occupancy are altered withinduced differentiation of VOT cells.

One hypothesis was that Pax2-Sox2 binding to the Atoh1 enhancer resultedin increased expression of Atoh1 by an effect on the modification ofhistones. To address whether interaction with DNA-binding co-factorsinfluenced bound histones at the Atoh1 locus, drugs that block histonemodifications were used. As shown in FIG. 15, treatment of OC-1 cellswith HDAC inhibitors both resulted in increased expression of Atoh 1.

These results demonstrate that modification of the epigenetic state ofAtoh1 can be used to upregulate expression of Atoh1, which is expectedto lead to generation of new hair cells in vivo.

Example 11. Epigenetic Modulation of Atoh1 by Hic1

Interactions of Sox2 and Wnt signaling pathways with the Atoh1 enhancersuggested the possibility that Sox2 and Wnt might act through epigeneticmechanisms. The present experiments test this hypothesis by determiningthe mechanisms through which Sox2, Pax2 and beta-catenin signalingaffect the epigenetic status of DNA.

Beta-catenin activities are modulated by Hic1, which interacts withpolycomb-like enzymes that methylate H3K27. Hic1 binds to andinactivates beta-catenin and Tcf4 (Valenta et al., EMBO J 25:2326-2337,2006), and methylation of the Hic1 gene leads to overactivity of Atoh1(Briggs et al., Cancer Res 68:8654-8656, 2008), which has been noted intumors of the cerebellum (Briggs et al., Genes Dev 22:770-785, 2008;Boulay et al., J Biol Chem 287:10509-10524, 2012).

Cochlear expression of Hic1 was assessed. Hic1 expression was not knownto be present in the cochlea. The P1 organ of Corti, where Sox2, Pax2and beta-catenin pathways are active showed strong expression of Hic1.

To address whether interaction with DNA-binding co-factors influencebound histones at the Atoh1 locus, inner ear stem cells were treatedwith Hic1 siRNA. As shown in FIGS. 17A-B, reducing Hic1 activity resultsin a dramatic increase in Atoh1 expression, and reducing Hic1 incombination with increased beta-catenin expression results in asynergistic increase in Atoh1 expression.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating sensorineural hearing lossassociated with loss of auditory hair cells in a subject, the methodcomprising administering a therapeutically effective amount of apharmaceutical composition comprising a histone methyltransferase (HMT)inhibitor to the subject having sensorineural hearing loss, wherein thetherapeutically effective amount is an amount sufficient to improvehearing at one or more frequencies.
 2. The method of claim 1, whereinthe HMT inhibitor is selected from the group consisting ofDeazaneplanocin A (DZNep), PR-SET7, GSK126, GSK J1, EPZ005687; E7438;EI1; EPZ-6438; GSK343; BIX-01294, UNC0638, BRD4770, EPZ004777, AZ505 andPDB 4e47.
 3. The method of claim 1, comprising administering thepharmaceutical composition to the inner ear of the subject.
 4. Themethod of claim 1, comprising application of the pharmaceuticalcomposition to the round window membrane.
 5. The method of claim 4,wherein the pharmaceutical composition is applied via intra-tympanicinjection or direct delivery into the inner ear fluids.
 6. The method ofclaim 5, wherein the direct delivery into the inner ear fluids isapplied using a microfluidic device.
 7. The method of claim 1, whereinthe pharmaceutical composition is formulated for administration to theinner ear of the subject.